1. Field of the Invention
The present invention relates to and has among its objects the provision of a novel method to obtain intact, viable plant protoplasts from within the cell walls of mature pollen grains.
2. Description of the Art
Pollen is unique among higher plant cells. It is haploid, that is, it has the number of chromosomes present in the normal reproductive cell which is equal to half the number in the normal somatic cell (cells that become differentiated and compose the tissues, organs, and parts of the individual plant). Each pollen grain is a single cell; it grows singly in culture behaving like a unicellular organism, unconfined by neighboring cells.
Pollen grains of flowering plants are usually spherical, elipsoid, or fusiform in shape. In its most basic form, a mature pollen grain has an inner protoplast cell surrounded by a protoplast membrane and an outer compound cell wall which is made up of an inner pectocellulosic layer, the intine, and an outer layer of sporollenin, the exine. The exine is characterized as a polymer of carotenoids and carotenoid esters and is highly resistant to biodegradation. One of the major functions of the cell wall is to prevent overexpansion, e.g., through uptake of water, of the protoplast with consequent rupture of the protoplast cell. Located in the cell wall are, in most cases, one or more small thinner walled areas, called pores, which represent positions of possible emergence of pollen tubes.
The protoplast represents the totality of living cell constituents. Protoplasts which have been obtained from within cell walls are capable of growing and dividing. Because of this, protoplasts have many important uses. One of the most important uses is the production of hybrid plants by somatic hybridization. For example, intra- and inter-generic or intra- and inter-specific plants can potentially be produced by hybridization. This is of particular value in those instances where sexual incompatibilities prevent such crosses by conventional pollination procedures. Other uses of isolated protoplast cells include: regeneration of new plants from a single cell; incorporation of genetic material into the cell; investigation of structural, physiological, and genetic alterations; elucidation of details of cell structure and function; and studies of plant virus infections and uptake of macromolecules by protoplasts.
In the isolation of plant protoplasts, an essential criterion is that release of the protoplast is obtained without causing irreversible damage to the protoplast. Of primary importance is the fact that protoplasts are osmotically active and will, under conditions of low or high osmotic potential, take up or lose water. When the cell wall (which exerts a wall pressure on the protoplast) is removed, it is critical that overexpansion and rupture of the cell caused by excessive uptake of water is prevented. Conventional methods to isolate protoplasts from plant tissue in general, e.g., leaf mesophyll cells, callus cells and the like include (1) mechanical cutting of the cell wall, (2) enzymatic degradation of the cell wall, and (3) use of solvents to dissolve the cell wall.
In the mechanical method, plant cells are placed in a plasmolyzing solution, e.g., calcium chloride or calcium nitrate (0.25 or 0.3M) or sucrose (1.0M), to shrink the protoplast away from the cell wall; then the wall is cut with a sharp knife. In some cases, a thin slice of the cell wall is cut and then the solution is changed to 0.5M sucrose to cause the protoplast to expand and force it through the cut surface. The major disadvantages of this method are that it is time consuming and the number of intact, undamaged protoplasts which can be removed by this by this method is few. Only those protoplasts in which marked plasmolysis and good separation of the protoplast from the cell wall occur can be successfully released from the cell wall using this method.
In the enzymatic method for the isolation of protoplasts, plant tissue, e.g., leaf mesophyll cells, are incubated with enzymes which free the cells, and the cells are treated with cell-degrading enzymes such as pectinase, cellulase, hemicellulase, or combinations thereof, to break down the cell wall and release the protoplast. While large quantities of released protoplasts can be obtained from leaf mesophyll tissue or callus tissue by this method, it has the disadvantage that substances in the crude enzyme preparation, e.g., proteases, lipases, perioxidases, and ribonucleases, can damage the protoplast or destroy the functional compounds in the protoplast membrace surface. Since the usefulness of the protoplasts is in their culture and further manipulation, it is essential that any adverse effect of the enzymes be minimized.
In the solvent method for the release of plant protoplasts, cell-degrading solvents, e.g., 4-methylmorpholine N-oxide monohydrate, are used to dissolve the cell wall. This is done at high temperatures (ca., 70.degree. to 80.degree. C.). The main disadvantage of this method is that damage to the protoplast can result from these strong solvents and the high temperature that has to be used.
With regard to mature pollen grains, the major difficulty encountered in release of protoplasts from the cell wall is the presence of the durable and highly resistant substance, sporopollenin, in the exine. Although, it is possible to partially degrade the exine through treatment with mixtures of cell wall-degrading enzymes, only limited quantities of protoplasts have been obtained by this method. Of major concern in the isolation of pollen protoplasts by this method is the potential harmful effects of the enzymes on the protoplasts. Mechanical isolation of protoplasts from pollen grains results in only limited numbers of protoplasts, and isolation of pollen protoplasts using solvent has the potential of damaging the protoplasts. In view of the potential usefulness of pollen protoplasts for use in somatic cell hybridization; regeneration of haploid plants; uptake of small and large molecules, viruses, bacteria, chloroplasts, DNA, and whole nuclei; and studies of mutation and cell modification, a method to readily obtain intact, viable protoplasts from within the cell walls of mature pollen grains is of great importance.